Chronic infection with HCV is a major health problem associated with liver cirrhosis, hepatocellular carcinoma and liver failure. Infection by HCV is insidious in a high proportion of chronically infected (and infectious) carriers who may not experience clinical symptoms for many years. Liver cirrhosis can ultimately lead to liver failure.
HCV is a member of the Flaviviridae family of RNA viruses that affect animals and humans. The genome is a single strand of RNA, and consists of one open reading frame that encodes for a polyprotein of about 3000 amino acids flanked by untranslated regions at both 5′ and 3′ ends (5′- and 3′-UTR). The polyprotein serves as the precursor to at least 10 separate viral proteins critical for replication and assembly of progeny viral particles. The organization of structural and non-structural proteins in the HCV polyprotein is as follows: C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b. While the pathology of HCV infection affects mainly the liver, the virus is found in other cell types in the body, including peripheral blood lymphocytes.
One treatment for chronic HCV involves interferon alpha (IFN-alpha) in combination with ribavirin and this requires at least six (6) months of treatment. However, treatment of HCV with IFN-alpha has frequently been associated with adverse side effects such as fatigue, fever, chills, headache, myalgias, arthralgias, mild alopecia, psychiatric effects and associated disorders, autoimmune phenomena and associated disorders and thyroid dysfunction. Ribavirin, an inhibitor of inosine 5′-monophosphate dehydrogenase (IMPDH), enhances the efficacy of IFN-alpha in the treatment of HCV. Despite the introduction of ribavirin, more than 50% of the patients do not eliminate the virus with the combination therapy of IFN-alpha and ribavirin.
Another therapy of chronic hepatitis C involves a combination of pegylated IFN-alpha plus ribavirin. However, a number of patients still experience significant side effects, primarily related to ribavirin. Ribavirin causes significant hemolysis in 10-20% of patients treated at currently recommended doses, and the drug is both teratogenic and embryotoxic. A substantial fraction of patients do not respond with a sustained reduction in viral load and there is exists a need for more effective antiviral therapy of HCV infection.
Other approaches for treatment include, for example, application of antisense oligonucleotides or ribozymes for inhibiting HCV replication. Furthermore, low-molecular weight compounds that directly inhibit HCV proteins and interfere with viral replication are potential strategies to control HCV infection. Among the viral targets, the NS3/4A protease/helicase and the NS5b RNA-dependent RNA polymerase are promising viral targets for new drugs.
One of the viral proteases, the NS3 protease, is encoded by the N-terminal region of the HCV NS3 gene. It consists of 181 amino acids and is a chymotrypsin-like serine-protease responsible for cleavage of the non-structural proteins of HCV [Bartenschlager et al., J. Virol. 67:3835-44.(1993)]. The hepatitis C virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor [Gallinari et al. Biochem Biophys Res Commun 192:399-406; (1998)]. Multiple enzymatic activities are associated with recombinant NS3 protein of hepatitis C virus [Hahm et al., J Virol 72:6758-6769 (1995)]. The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A. [Tanji et al., J Virol 69:4255-4260 (1995)]. Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing [Tanji et al., J Virol:1575-1581)]. In addition, NS3 contains a tetrahedrally bound zinc atom, which appears to play a structural role [De Francesco et al., Biochemistry 35:13282-7 (1996)].
Models for how the protease interacts with cofactors and the substrate have identified four domains which are involved in enzyme function [Barbato et al. J. Mol. Biol., 289, 371-384 (1999). The solution structure of the N-terminal proteinase domain of the HCV NS3 protein provides new insights into its activation and catalytic mechanism. [Id.]. The activation and catalytic mechanism of the enzyme appears to involve a catalytic triad, cofactor and metal binding sites and the substrate-binding pocket.
The compounds described below are potent inhibitors of the NS3 protease and are useful for inhibiting viral replication in vitro and in vivo. The compounds are useful for treating HCV infection in subjects, particularly human subjects.